The present invention relates to chiral cis-azetidinones having an unsaturated side-chain prepared by enantioselective acylation of a monocyclic xcex2-lactam with Penicillin G Amidase.
Non-classical xcex2-lactams such as the monobactams (e.g., aztreonam), carbapenems (e.g., thienamycin), and carbacephems (e.g., loracarbef) have been shown to be clinically effective anti-bacterial agents. Much attention has been focussed on the development of economical, large scale synthesis of these derivatives. Since loracarbef is available only via a total synthesis, improvements in the manufacturing process are of particular interest. One of the most challenging synthetic steps is the resolution of the chiral cis-xcex2-lactam intermediates. U.S. Pat. No. 5,057,607 provides a enantiomerically selective biocatalyzed acylation of a racemic mixture of cis-3-amino-2-[2-(2-furanyl)ethyl]-4-oxo-1-azetidine acetic acid (or its alkyl ester) with methyl phenylacetate (or methyl phenoxyacetate) in the presence of a penicillin G amidase enzyme. The racemic materials serve as the substrate for the enzyme. Once acylated, the acylated active enantiomer is isolated and the inactive enantiomer is disposed of thus reducing the overall yield of the intermediate. Therefore, there is a need for a means of producing the desired enantiomeric cis-xcex2-lactam intermediate with higher overall yields.
The present invention provides a cis-xcex2-lactam compound having structure II and a process for preparing the cis-xcex2-lactam compound having structure II: 
where R1 is xe2x80x94SO3xe2x88x92, xe2x80x94SO3(C1-C4)alkyl, xe2x80x94CH2CO2H, or xe2x80x94CH2CO2R1xe2x80x2, where R1xe2x80x2 is (C1-C4)alkyl, benzyl, or substituted benzyl (e.g., p-nitrophenylmethylene); R2 is benzyl or phenoxymethylene; and R3 is 2-furyl, phenyl, or 2-methoxyphenyl. In a preferred embodiment, R1 is xe2x80x94CH2CO2H or an ester thereof. Compound II is prepared by the steps of
(i) providing a racemic mixture of cis-azetidinones having structures Ia and Ib 
xe2x80x83where R1 and R3 have the same meaning as above, and (ii) reacting the racemic mixture with a (C1-C4)alkyl phenylacetate or a (C1-C4)alkyl phenoxyacetate in the presence of penicillin G amidase. Once acylated, Compound II may be further modified. For example, Compound II may be hydrogenated to form a compound having a saturated sidechain (Compound III below). 
As used herein, the term xe2x80x9c(C1-C4)alkylxe2x80x9d refers to an alkyl group having one to four carbon atoms (e.g., methyl, ethyl, propyl and butyl). The alkyl group may be a straight or branched chain (e.g., n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, and t-butyl)
The use of Penicillin G Amidase for the enzymatic acylation of xcex2-lactam antibiotics and resolution of cis racemic azetidinones has been previously reported. (see, e.g., U.S. Pat. No. 5,057,607, incorporated herein by reference; Zmijewski Jr., M. J., et al., xe2x80x9cEnantioselective acylation of a Beta-Lactam Intermediate in the synthesis of Loracarbef using Penicillin G Amidase,xe2x80x9d Tetrahedron Letters, 32(13), 1621-1622 (1991); and Briggs, B. S., et al., xe2x80x9cSide Chain Selectivity and Kinetics of Penicillin G Amidase in Acylating a Cis-Racemic xcex2-Lactam Intermediate in the Synthesis of Loracarbef,xe2x80x9d New J. Chem., 18, 425-434 (1994).) However, none of the previous investigators realized the utility and advantages of enantioselectively acylating a cis-xcex2-lactam intermediate having an unsaturation in the side chain. Since enzymes are very substrate sensitive, it was also unknown until the discovery by the Applicants that one could enantioselectively acylate a cis-xcex2-lactam intermediate having an unsaturation in the side chain in the presence of Pencillin G Amidase.
The presence of the unsaturation in the side-chain provides several advantages that cannot be realized in the previously disclosed hydrogenated intermediate. For example, one may be able to recycle the undesired inactive cis-enantiomer by racemizing the unwanted cis-isomer. Scheme I below illustrates a potential means of providing this racemization and subsequent enantioselective acylation to increase the overall yield of the desired cis-enantiomer. 
Applicants have observed that when the methyl ester of cis-3-amino-2-[trans-2-(2-furanyl)ethylene]-4-oxo-1-azetidineacetic acid is converted to its 3-p-nitrobenzyl imine derivative under basic conditions epimerization of the C-3 stereocenter occurs. Other substituents located alpha to the C-3 and/or C-2 stereocenters have also been shown to activate azetidinones towards racemization. (see, e.g., Alcaide, B., et al., Tetrahedron Letters, 39, 5865-5866 (1998); Suarato, A., et al, Tetrahedron Letters, 42, 4059-4062 (1978); and Kametani, T., et al, Heterocycles, 16(4), 539-547 (1981)). Therefore, it is reasonable to believe that treatment with base will epimerize the two chiral centers thus providing a means for recovering additional desired enantiomer. Suitable bases include organic bases (e.g., trialkyl amines, pyridines, pyrimidines, quinolines, isoquinolines and derivatives thereof), metal alkoxides, diazabicyclo[5.4.0]undec-7-ene (DBU). Generally, the reaction is run in a non-nucleophilic solvent (e.g., methylene chloride, ethers, hydrocarbons, benzene and toluene) at about xe2x88x9235xc2x0 C. to about 70xc2x0 C.
The presence of the unsaturation in the azetidinone also provides a reactive site for derivatization. For example, the unsaturated side-chain may be converted to a methyl group. (see Scheme II) Cleavage of an unsaturation to produce an alkyl group is well known to those skilled in the art. The enantioselective acylation followed by conversion of the unsaturated side-chain could provide an alternative method for making enantiomerically pure intermediates in the synthesis of monobactams (e.g., Azetreonam). 
Alternatively, one could oxidatively cleave the unsaturation to provide a carboxyl or aldehyde group at the 2 position of the xcex2-lactam ring. The oxidized product could then be derivatized to provide a variety of intermediates that could be used in the development of new antibiotic agents.
The racemic mixture of cis-xcex2-lactams can be obtained by methodology well-known to those skilled in the xcex2-lactam art. For example, the cis-xcex2-lactam racemates may be produced using the ketene/imine (2+2) cycloaddition reaction described in Hatanaka, et al., Tetrahedron Letters, 24(44), pp. 4837-4838 (1983); Georg, F. I. and V. T. Ravikumar, The Organic Chemistry of xcex2-Lactams, Chapter 6, G.I. Georg (Ed.), VCH, pp 295-368 (1993); and U.S. Pat. Nos. 4,260,743 and 5,159,073, both incorporated herein by reference. A preferred method utilizes the formation of a xe2x80x9cDane saltsxe2x80x9d which is formed by allowing a solution of a potassium salt of an xcex1-amino acid to react with a xcex2-dicarbonyl compound. The Dane salt is then reacted with ethyl chloroformate and, triethylamine to form a mixed anhydride which is then reacted in situ with a Schiff base to afford the cis-isomer stereoselectively. For a more detailed description, see the preparation section of the Examples.
The term xe2x80x9cpenicillin G amidasexe2x80x9d (or the alternative term xe2x80x9cpenicillin G acylasexe2x80x9d) is well-known in the xcex2-lactam art as an enzyme which catalyzes the hydrolysis of the penicillin G sidechain(phenylacetyl) from penicillin substrates. Penicillin G amidases suitable for use in the process of the present invention may be isolated by known methodology from many organisms, for example, E. coli, B. megaterium, Ps. melanogenum, K. citrophila, and P. rettgei. In this regard Schlwale and Sivarawan, Process Biochemistry, August., 1989, pp. 146-154 sets forth a review of the state of the art of penicillin G amidase (acylase) production and application. Penicillin G amidase isolated from E. coli is preferred.
Once isolated, the penicillin G amidase may be used in xe2x80x9cfreexe2x80x9d form, i.e., solubilized in aqueous or substantially aqueous solutions, or may be immobilized onto a support matrix such as an intermolecular adduct with glutaraldehyde; Sepharoses; Sephadex G-200(trademark), acrylamide, N,N-methylenebis (acrylamide) and maleic anhydride; Dextran(trademark); maleic anhydride; tetramethyleneglycol; dimethacrylate; methacrylic acid, DEAE-Cellulose(trademark); CM-Cellulose(trademark); AE-Cellulose(trademark); and other cellulose derivatives; CM-Sephadex Amberlite IRC-50(trademark). and other weak cation and anion exchangers; ethylene maleic anhydride copolymers; Nylon(trademark); Amberlite XAD-7(trademark); Sucrose/epichlorohydrin copolymer; polyacrylamide; cellulose; intermolecular adduct with glutaraldehyde; acrylamide copolymer; anion exchange phenol-formaldehyde resin; DEAE-Sephadex(trademark); glycidyl methacrylate; methylene bisacrylamide; diatomaceous earth; poly(hydroxyethyl methacrylate); Eupergit C(trademark); basic anion exchanger (polyamine; styrene; divinylbenzene); cellulose triacetate fibres; AH-Sepharose(trademark)/benzoquinone; nitrocellulose fibres; a polyethylene imine; Bentonite(trademark); a polyacrylamide gel entrapment or derivatised polyacrylonitrile.
The immobilized penicillin G amidase may be obtained commercially. For example, the immobilized enzyme used in the experimental section below was obtained from SCLAVO S.p.A.xe2x80x94Biochemical Division De. Bi., S. S. Podana Superiore, Km. 160,20060xe2x80x94Cassina de Pecchixe2x80x94Milan, Italy. It is believed that any penicillin G amidase enzyme will be efficacious as a biocatalyst in the present invention whether used in free form or immobilized on a support matrix; however, it is preferred that the enzyme be immobilized on a solid support matrix, because such catalysts can be used several times. For example, when the reaction is deemed complete, the immobilized enzyme may simply be filtered away from the reaction mixture, washed with deionized water, stored in glycerol/water under an inert atmosphere such as nitrogen or argon at reduced temperature, for example at about 4xc2x0 C., and re-used.
The substrate is preferably present in the reaction mixture in a concentration of 0.1% (w/w) to about 20% (w/w) although concentration is not critical to the operability of the process. The amount of penicillin G amidase present in the reaction mixture dictates the rate of reaction, because it serves as a biocatalyst. A concentration of from about 10 I.U./g substrate (i.e., the xcex2-lactam of formula (2)), to about 125 I.U./g substrate of penicillin G amidase is preferred. More preferably, the concentration will be at the lower end of the foregoing range, i.e., from about 10 I.U./g substrate to about 30 I.U./g substrate, and, most preferably, from about 15 I.U./g substrate to about 25 I.U./g. In the context it is used herein, one international unit (I.U.) is the amount of enzyme that will catalyze hydrolysis of one xcexcmole of penicillin G in one minute at 28xc2x0 C.
The acylation reaction of the present invention may be carried out in aqueous media at a pH of about 5 to about 8, preferably at about pH=6, thus providing an environmentally-compatible synthesis of intermediates useful in the synthesis of 1-carba(dethia)3-cephems which is suitable for use in large scale synthesis. Alternatively, the reaction may be carried out in a water/water-miscible polar organic solvent mixture comprising from about 1 to about 28% of a polar organic solvent such as acetone, tetrahydrofuran, propylene glycol methyl ether, propylene glycol, ethylene glycol dimethyl ether, 2-methoxyethyl ether, ethylene glycol, or glycerol, and from about 99% to about 72% water.
The temperature at which the process may be carried out will be appreciated by one of ordinary skill in enzyme catalysis and thus is not a critical limitation of the process; however, a temperature range of about 10xc2x0 C. to about 45xc2x0 C. is preferred. A more preferred temperature is about 28xc2x0 C.
Once acylated, the vinyl side-chain may be hydrogenated. A standard hydrogenation is accomplished in the presence of a hydrogenation catalyst and a solvent, such as methylene chloride, dimethylformamide, ethers, esters and mixtures thereof. Suitable hydrogenation catalysts include nickel, platinum, rhodium, ruthenium, copper chromite, iridium, osmium, palladium, and combinations thereof. A preferred catalyst is a supported palladium, e.g., 5% or 10% palladium on carbon, barium carbonate, or other suitable support. The reduction generally is carried out at atmospheric conditions or at somewhat elevated pressures, and at substantially room temperature.